Anthrax is caused by infection with Bacillus anthracis, a spore-forming, rod-shaped bacterium. The dormant spore-form is highly resistant to extreme conditions, high temperatures, and a variety of chemical treatments. The spores gain entry either through an open wound, causing cutaneous disease, or by ingestion, causing gastrointestinal disease or are inhaled causing inhalation anthrax. All three forms can progress to a systemic infection leading to shock, respiratory failure, and death. (Mock, M. and Mignot, T. (2003), Cell Microbiol., 5(1):15-23). The stability of the spores and their infectious capacity make them a convenient bioterrorist weapon.
The two known toxins of B. anthracis are binary combinations of protective antigen (PA), named for its ability to induce protective immunity against anthrax, with either edema factor (EF) or lethal factor (LF). PA is the cell binding component of both toxins and is responsible for bringing the catalytic EF or LF into the host cells. EF is an adenylate cyclase that converts ATP to cyclic AMP and causes edema (Brossier, F. and Mock, M. (2001), Toxicon. 39(11):1747-55). The combination of PA-EF forms edema toxin (ETx) which causes edema when injected locally. LF is a zinc-dependent endoprotease known to target the amino-terminus of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators (Id.). The cleavage of these proteins disrupts a signaling pathway and leads to cytokine dysregulation and immune dysfunction. LF combined with PA forms lethal toxin (LTx), which is lethal when injected on its own. It is also known that there are fatal anthrax cases where administration of antibiotics and clearance of bacteria have failed to rescue the patient. This indicates that there may be a “point of no return” level of LTx in the blood that may predict the outcome of infection. Clearly, LTx and its components are important targets for diagnostics and quantification.
Assays for EF activity such as competitive enzyme assays (Duriez, E, et al., Anal. Chem., 2009; 81:5935-5941) or radiometric assays (Gottle, M, et al., Biochemistry, 2010; 49:5494-503), are impractical for high-throughput screening of compound collections and rapid diagnosis of host infection. Methods for rapid screening of patients in a hospital setting or identification of potent and selective EF inhibitors requires an assay that is less labor intensive, has faster turnaround, and is effective at low levels of enzyme.
Development of targeted therapies following anthrax infection is essential to managing a patient population. As such, there exists a need for compositions and methods that can be used as aids in a screening assay for identification and development of inhibitors of anthrax toxin activity or for identifying active LF such as in the circulation of a subject.